Journal of
Plankton Research
plankt.oxfordjournals.org
J. Plankton Res. (2015) 37(5): 897– 911. First published online August 28, 2015 doi:10.1093/plankt/fbv066
LEANNE E. ELDER†* AND BRAD A. SEIBEL
DEPARTMENT OF BIOLOGICAL SCIENCES, UNIVERSITY OF RHODE ISLAND,
120 FLAGG ROAD, KINGSTON, RI 02881, USA
†
PRESENT ADDRESS: DEPARTMENT OF GEOLOGY AND GEOPHYSICS, YALE UNIVERSITY, NEW HAVEN, CT USA
*CORRESPONDING AUTHOR: E-mail leanne.elder@yale.edu
Received January 11, 2015; accepted July 26, 2015
Corresponding editor: Marja Koski
Phronima sedentaria is a hyperiid amphipod that diel migrates into a pronounced oxygen minimum zone (OMZ) in the
Eastern Tropical North Pacific. In this study, oxygen consumption and lactate production were measured in P. sedentaria
to estimate the aerobic and anaerobic contributions to total metabolism under conditions that mimic its day- (1%
oxygen, 108C) and night-time (20% oxygen, 208C) habitat. When exposed to hypoxia and low temperature, the total
metabolism of P. sedentaria was depressed by 78% compared with normoxic conditions. The metabolic enzymes citrate
synthase (CS) and lactate dehydrogenase (LDH) were also measured as indicators of aerobic and anaerobic metabolism, and compared with specimens collected from the California Current and the North Atlantic to assess potential
adaptations to low oxygen. LDH activity was not significantly different between regions. Significant differences in CS
activity may be due to variation in food availability. Climate change is predicted to increase surface temperatures and
cause the expansion of OMZs. This will result in vertical compression of the night-time range for P. sedentaria and is
likely to have the same impact on other diel migrators. Habitat compression will reduce zooplankton contribution to
carbon cycling and alter oceanic ecology, including predator– prey interactions.
KEYWORDS: metabolic depression; climate change; hypoxia; anaerobic metabolism; hyperiid amphipods
I N T RO D U C T I O N
In some regions of the oceans at intermediate depths,
biological oxygen use exceeds the rates of oxygen replenishment via the processes of advection and diffusion
(Packard et al., 1988) leading to zones of low oxygen.
These oxygen minimum zones (OMZs) occur in areas of
high primary productivity such as the Eastern Tropical
North Pacific (ETNP), where organic matter from the
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Ecophysiological implications of vertical
migration into oxygen minimum zones for
the hyperiid amphipod Phronima sedentaria
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the sum of aerobic and anaerobic metabolism) to limit the
accumulation of harmful anaerobic end products (e.g. Hþ)
and to conserve fuel stores (Seibel, 2011). Metabolic depression (also known as metabolic suppression) is a common response among marine animals to environmental stressors
such as desiccation, food deprivation, and low oxygen
(Storey and Storey, 1990; Guppy and Withers, 1999).
Hyperiid amphipods are the third most abundant
crustacean marine zooplankton, after euphausiids and
copepods (Diebel, 1988). Phronima sedentaria (Fig. 1), in
particular, has a worldwide distribution (Shih, 1969) and
is abundant in the pronounced OMZ of the ETNP.
Phronima sedentaria is a diel vertical migrator, spending
night time near the surface (0 – 25 m) and living as deep
as 600 m during the day (exact maximum depth is not
known, but is between 300 and 600 m) (Shih, 1969;
Shulenberger, 1977). Like most hyperiid amphipods,
P. sedentaria often lives parasitically on tunicates or siphonophores, using them as a food source and a brood
chamber (Madin and Harbison, 1977; Laval, 1978).
Phronimids eat the internal tissue of their host leaving
the remaining gelatinous matrix in a barrel shape (Hirose
et al., 2005) that is propelled through the water with the
urosoma (tail) half out the back (Land, 1992). Childress
and Seibel (Childress and Seibel, 1998) suggested that
amphipods may be especially tolerant of low oxygen
because their gelatinous host provides a substrate that
can fuel extended anaerobic metabolism.
This study was conducted to determine whether, and
to what extent, P. sedentaria depresses metabolism to
survive migration into a pronounced OMZ and how
much it relies on anaerobic metabolism. To test this, total
metabolism was estimated from the accumulation of
anaerobic end-products and the rates of oxygen
Fig. 1. Phronima sedentaria from the Eastern Tropical North Pacific. This
individual has been accessioned to the Yale Peabody Museum (YPM) as
a taxonomic voucher for other specimens used in the study. It is
catalogue number YPM IZ 075000. Scale bar equals 5 mm.
Photograph taken by Eric Lazo-Wasem.
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surface sinks and decays, adding to the oxygen demand
at intermediate depths (Fiedler and Talley, 2006). The
OMZ in the ETNP is remarkable for both its size and
degree of hypoxia (Kamykowski and Zentara, 1990).
This OMZ extends vertically from 50 to 1200 m
(Fernández-Álamo and Färber-Lorda, 2006). Below
300 m oxygen levels vary, but can be ,2 mM (0.15 kPa,
0.04 mL L21) (Wishner et al., 2013). The California
Current has a less severe OMZ, with oxygen levels reaching a minimum of 13.4 mM (0.8 kPa, 0.3 mL L21)
(Childress and Seibel, 1998).
OMZs are predicted to expand both vertically and
horizontally as a result of the changing world climate
(Stramma et al., 2008; Keeling et al., 2010; Deutsch et al.,
2011). Most of the oxygen decrease is attributed to
increased stratification, which limits the mixing of oxygenated surface waters with subsurface waters and reduces
the subsurface oxygen concentrations (Keeling and
Garcia, 2002). Increasing global temperatures will warm
ocean surface waters, leading to a decrease in oxygen
content due to decrease in oxygen solubility. Oxygen
levels influence vertical distribution and ecology of
marine animals (Vinogradov et al., 1996; Wishner et al.,
2013). Understanding how oxygen concentrations affect
zooplankton physiology is important because expanding
OMZs may cause alterations in species’ vertical and horizontal habitat ranges. Those alterations could, in turn,
change ecosystem trophic structures due to shifts in
predator– prey interactions as well as affecting carbon
cycling (Seibel, 2011; Doney et al., 2012).
Most studies on hypoxia tolerance of marine animals
have been conducted in OMZs where dissolved oxygen
levels are relatively higher than in the OMZ of the
ETNP. Organisms found in the California Current OMZ
are often able to remain aerobic (Childress, 1977). This
ability to extract oxygen from hypoxic water is due to
adaptations including: increased ventilation and circulation capacity, high gill surface area, short blood to water
diffusion distances and respiratory proteins with high
oxygen affinity and cooperativity (Childress and Seibel,
1998). In moderate OMZs, the majority of the biomass is
permanent deep-living zooplankton and micronekton
throughout the depth range (Vinogradov et al., 1996;
Childress and Seibel, 1998; Robinson et al., 2010).
At oxygen concentrations less than 10 mM in the
ETNP, there is a reduction in biomass at depth. Most
organisms either live at the upper or lower OMZ interfaces (zones of steep oxygen gradients), or vertically
migrate to more oxygenated waters at night (Vinogradov
and Voronina, 1962; Wishner et al., 1990, 2013).
However, organisms accustomed to variable and transient hypoxia, such as that experienced by diel vertical
migrators, will often depress their total metabolism (i.e.
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consumption under hypoxia and normoxia. Metabolic
enzyme activities were also measured as indicators of the
capacity for aerobic and anaerobic metabolic rate in P.
sedentaria from regions with varying oxygen levels.
METHOD
Collection
Metabolic rate (MO2)
After collection, parasitic specimens were gently removed
from their host. Specimens were individually transferred
to filtered seawater within a half hour of collection and
held in a water bath at experimental temperature for at
least 12 h, ensuring they were acclimated and starved.
Filtered (0.2-mm demicap filter, Fisher Scientific, USA)
and treated (25 mmol L21 each of streptomycin and ampicillin) seawater was prepared for respiration chambers
Fig. 2. Representative water profiles of the top 500 m for all study locations. (A) Temperature profiles and (B) oxygen profiles. Data were collected
with shipboard conductivity, temperature, density (CTDs). Black dashed line: North Atlantic, 39858N, 67859W, 25 September 2011. Dark grey solid
line: ETNP St 1, Eastern Tropical North Pacific Station 1, the Tehuantepec Bowl, 118N 988W. Black solid line: ETNP St 2, Eastern Tropical North
Pacific Station 2, the Costa Rica Dome 8.58N 908W, 2 January 2009. Light grey dashed line: Gulf of California, 27814N 111829W, June 2007. Light
grey solid line: California Current, 33844N, 118846W, 11 November 2012.
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Specimens of P. sedentaria were collected from the ETNP,
the Gulf of California and the North Atlantic (see
Supplementary data, Table SII for precise locations) using
a modified Tucker trawl equipped with a 30-L thermally
insulated cod-end (Childress et al., 1978). The net was
opened and closed using a MOCNESS-type step motor
and equipped with temperature and pressure sensors.
Specimens from the California Current were collected
using a 505-mm mesh bongo net and a 1-m2 MOCNESS
net with 332-mm mesh. A conductivity, temperature,
density (CTD) cast was conducted daily at each station to
obtain water profile information (Fig. 2). For all locations,
the majority of net tows were done at night (between 1900
and 2400 local time) from 20 to 50 m. Some specimens
were collected from the North Atlantic and California
Current during the day (1300 local time) at depths
between 350 and 250 m. Only female specimens were
used for this study because they were more abundant than
males in all locations (see Supplementary Material online
for details on sex ratio).
Specimens from each location were used for metabolic
rate experiments. Enzyme activities were compared
between specimens collected from: the ETNP, a region with
a pronounced OMZ; the California Current, where the
oxygen levels are higher than in the ETNP; and the North
Atlantic, which does not have a strong OMZ (Fig. 2B).
To compare environmental lactate production with
laboratory experiments, a field study was conducted.
Phronima sedentaria specimens were collected in two separate
trawls, one within and one above the OMZ, during the
day and night, respectively. These trawls were done on 2
January 2009 in the ETNP (Fig. 2, see Supplementary
data, Table SII). The deep trawl was 250–300 m and the
shallow trawl was 25–50 m. Upon net retrieval, 10 individuals all alive and in good condition were immediately
frozen in liquid nitrogen and transferred to a 2808C
freezer. Specimens were weighed in the laboratory prior to
L-lactate measurement.
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consumption rates were determined by calculating the
difference in final oxygen concentration between the
control and experimental chambers and incorporating
the adjusted volume of water, mass of the organism and
time elapsed. At the end of incubations, specimens were
immediately blotted dry, frozen in liquid nitrogen, then
transferred to a 2808C freezer. Weights were determined
from frozen individuals in the laboratory for all specimens
except for those collected in the Gulf of California, which
were weighed on a shipboard balance system (Childress
and Mickel, 1980) and frozen in liquid nitrogen. Metabolic rate was determined per hour incubation per gram
body weight for each individual.
A temperature coefficient, or Q 10 ð¼ðR2 =R1 ÞððT2 T1 Þ=10Þ,
R ¼ oxygen consumption rate, T ¼ temperature, quantifies the factorial change in metabolic rate with 108C
change in temperature and typically falls in the range of
2 – 3 (Hochachka and Somero, 2002). Q 10 was calculated
from the average mass-specific routine metabolic rate at
each temperature and used to normalize metabolic rates
to 208C for comparison (Supplementary data, Fig. S2
and Table SI).
L-Lactate
measurements
To determine reliability of handheld lactate metres, measurements of lactate standards were compared using the
traditional spectrophotometric method (Gutmann and
Wahlefeld, 1974; Engel and Jones, 1978), and the lactate
metres: Accutrend (Roche Diagnostics Corp., Indianapolis,
USA), and lactate plus (Nova Biomedical, USA). Using
the metre instead of the spectrophotometric method
reduces cost and duration of sample processing. In the
preliminary trials for this study, the lactate plus metre
was not sensitive to lactate values ,10 mmol g21. The
Accutrend lactate metre provided measurements comparable with the spectrophotometric method. Other
studies have also demonstrated that the Accutrend metre
is an acceptable alternative to the spectrophotometric
method for lactate measurement (Beecham et al., 2006;
Pérez et al., 2008).
Lactate was measured in whole organisms from the
ETNP. Tissue-specific measurements would miss lactate
present in other parts of the body. Determining lactate of
the whole organism allows lactate involved in exchange
mechanisms, known as lactate shuttles (Brooks, 2002), to
be accounted for. Measurements were done on the same
specimens used for oxygen consumption in order to calculate the total metabolism for each individual.
Whole frozen specimens were ground on ice in a prechilled glass tissue homogenizer (Kimble Chase, USA) using
a 1:2 or 1:1 dilution with homogenization buffer (465 mm
NaCl, 19 mm KCl, 20 mm Tris). The homogenate was
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with a water-jacketed gas-equilibration column, which was
connected to a temperature-controlled water bath (Lauda,
Germany) set to the desired experimental temperature
(10, 15, 20 or 258C). For hypoxic experiments (only
performed in the ETNP), the water column was bubbled
with a certified gas mixture of 1% oxygen (10 mM,
0.8 kPa at 108C). For normoxic experiments, water was
bubbled with 21% oxygen, (balanced with nitrogen) to
ensure air saturation. Experimental water was bubbled for
a minimum of 1 h and checked with a Clark-type oxygen
electrode (Clark, 1956, described below), to ensure it
reached the desired oxygen concentration. Hypoxic treatments were conducted at 10, 15 and 208C. Normoxia
treatments were conducted at 10, 15, 20 and 258C in the
ETNP and 10, 15 and 208C in the Gulf of California.
Hypoxia at 108C is consistent with conditions in the
ETNP at 300 m depth, 158C is the temperature at intermediate depths of P. sedentaria’s distribution, and 208C is
the temperature experienced at night. Surface temperatures can reach 258C in the ETNP, so P. sedentaria may
occasionally experience temperatures that high.
Depending on the size of the organism, either 25-mL
glass scintillation vials or glass gas-tight syringes were
used as respiration chambers. There was no significant
difference in metabolic rate between the chambers used
for hypoxic (unpaired t-test: t (17) ¼ 1.06; P ¼ 0.30) or
normoxic conditions (t (66) ¼ 1.74; P ¼ 0.09). Chambers
were filled with water from the gas-equilibration column
and an individual was immediately placed in the
chamber. A blank chamber with no specimen was filled
with identically treated water and processed simultaneously to monitor background respiration of microbes.
The chambers were sealed (air bubbles removed) and
incubated in a temperature-controlled water bath (Lauda,
Germany). All experiments were carried out in darkness.
Normoxia experiments were conducted for 5–27 h. The
size of individuals was used to estimate the duration
needed to provide measureable changes in oxygen saturation. Hypoxia experiments were incubated for a shorter
duration of 2–6 h to prevent complete depletion of
oxygen in the chambers. Metabolic rate was calculated as
per gram per hour to normalize for size and duration.
Water was removed from incubation chambers using a
500-mL syringe (Hamilton, USA). Oxygen concentrations
were then measured using a Clark-type oxygen electrode
(Clark, 1956) connected to a Strathkelvin Instruments 782
Oxygen Interface (Strathkelvin Instruments, UK). The
oxygen electrodes were maintained in a thermally jacketed
electrode holder (MC100 Microcell, Strathkelvin Instruments) attached to the water bath of the appropriate
experimental temperature (Marsh and Manahan, 1999).
The electrode was calibrated prior to measurements
using air- and nitrogen-saturated seawater. Oxygen
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centrifuged at 2000 rpm for 5 min at 48C and the supernatant was removed. L-Lactate concentrations were measured on the Accutrend lactate metre using 25 mL of
supernatant. All samples were assayed in triplicate, and compared with a lactate standard curve (sodium lactate, L7022,
Sigma-Aldrich, MO, USA) which was run daily. The
Accutrend lactate metre measures lactate using enzymatic
determination and reflectance photometry at a wavelength
of 660 nm (Beecham et al., 2006).
Total metabolism
Enzymatic activity
Live specimens were identified and flash frozen in liquid
nitrogen at sea. Frozen specimens were shipped back to
the University of Rhode Island on dry ice and stored at
2808C. Metabolic enzymes citrate synthase [CS,
Enzyme Commission number (EC) 4.1.3.7] and lactate
dehydrogenase (LDH, EC 1.1.1.27) were measured.
Individual, frozen P. sedentaria were homogenized on
ice in 0.01 M Tris buffer, ( pH 7.5 at 108C) in a prechilled glass tissue homogenizer (Kimble Chase) using a
1/3 dilution for CS and a 1/3 – 1/15 dilution for LDH
(depending on size and activity levels). Homogenate was
centrifuged at 48C, 4500 rpm for 10 min. Aliquots of
supernatant (25 mL) were added to 1-mL cocktail solution in a quartz cuvette. Assays were performed at 208C
using a spectrophotometer (UV160 U, Shimadzu Scientific instruments, Japan) equipped with a water-jacketed
cuvette holder connected to a recirculating water bath.
Statistics
Statistics were performed using the software SAS version
9.2 (SAS Institute, Inc., USA). One-tailed Student’s t-tests
were used to compare metabolic rates scaled to a common
body size. One-way analysis of variance (ANOVA) and
one-way analysis of covariance (ANCOVA) were used to
compare differences between treatments.
Linear regression was used to test the relationship
between body mass and metabolic rate. Mass-specific
metabolic rate (MO2) and enzymatic activities typically
decline with increasing body mass (M) according to a
power equation (MO2 ¼ aMb), where a is a normalization
constant, and b is a scaling coefficient, which describes the
slope of the relationship. The relationships of metabolism
and enzymatic activities versus mass were linearly
regressed on a log scale using KaleidaGraph version 4.1
(Synergy Software, USA) to obtain the power equation.
R E S U LT S
Metabolic rate
In the ETNP, the average oxygen consumption for
P. sedentaria normalized to 208C was 3.65 + 0.26 mmol
O2 g21 h21 in normoxia and 1.87 + 0.73 mmol O2 g21 h21
in hypoxia (Table I). MO2 from ETNP specimens was
plotted on a log axis to obtain regression equations and
was significantly related to body mass for hypoxic and
normoxic treatments (Fig. 3). MO2 was significantly
related to body mass according to MO2 ¼ 0.3268 901
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Glycogen stores have been shown to be an important
energy store in gammariid crustaceans (Foucreau et al.,
2013). Assuming glycogen stores are also used by hyperiids
amphipods as substrate during anaerobic metabolism, 1.5
mol of adenosine triphosphate (ATP) are produced per
mole lactate accumulated. Six moles of ATP are produced
per mole O2 consumed during aerobic metabolism
(Mcdonald et al., 1998). Combining these components provides a measure of the total ATP produced (total metabolism). Lactate is formed and utilized under fully aerobic
conditions for cell signalling and delivery of substrates
(Brooks, 2002). Therefore, lactate produced in normoxia is
considered to be the stable pool of lactate for an organism’s function. The amount of ATP produced from this
stable pool of lactate was calculated in normoxia for each
individual and then the average amount for each temperature (10 or 208C) was subtracted from the total ATP produced in normoxic and hypoxic conditions at the same
temperature. Metabolic depression was then calculated
from the reduction in total ATP produced when exposed
to hypoxic conditions.
Supernatant was kept on ice until measurements, which
were done within 1 h of homogenization in triplicate
when possible (some specimens were too small to allow
for this). Activities are expressed as mmol of substrate
converted to product min21 g21 frozen tissue weight.
The CS cocktail solution is made of: 0.05 M imidazole
buffer, 15 mM magnesium chloride, 4 mM 5,5-dithiobis-2-nitronezoic acid (DTNB) and 3 mg acetyl coenzyme A. 25 mL of 40 mM oxaloacetate was added to
start the reaction. The background activity was measured
before the addition of oxaloacetate and subtracted from
the final rate to derive CS activity. The spectrophotometer measures the increase in absorbance at 412 nm,
which follows the increase of absorbance as Coenzyme A
is reduced by DTNB (Gutmann and Wahlefeld, 1974).
The LDH cocktail solution is made of: 0.2 M Tris ( pH
7.2 at 208C), 0.15 mM NADH, 100 mM KCl, 0.5 mM
Na-pyruvate and distilled water. The spectrophotometer
records the oxidation of NADH through the decrease in
absorbance at 340 nm (Bergmeyer et al., 1985).
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Table I: Routine mass-specific oxygen consumption rates (mmol O2 g21 h21) in Phronima sedentaria
under normoxia and hypoxia in the three study locations
Treatment
MO2 (mmol O2 g21 h21)
Location
N
Regression equation
Normoxia
Hypoxia
Normoxia
Normoxia
3.65 + 0.26
1.87 + 0.73
2.99 + 0.155
6.34 + 0.94
Eastern Tropical North Pacific
Eastern Tropical North Pacific
Gulf of California
North Atlantic
39
19
49
4
MO2 ¼ 2.4572 M (20.2079) R 2 ¼ 0.21
MO2 ¼ 0.3268 M (20.543), R 2 ¼ 0.58
MO2 ¼ 1.907 M (20.25), R 2 ¼ 0.29
MO2 ¼ 3.92 M (20.263), R 2 ¼ 0.93
L-Lactate
The concentrations of L-lactate in whole organism
samples of P. sedentaria, after 5 h of exposure to 1%
oxygen or normoxia levels at different temperatures, are
presented in Fig. 4. Total L-lactate concentrations in
whole organisms were significantly higher (t (34) ¼ 24.76;
P , 0.0001) in hypoxic (10.49 + 1.82 mmol g21, n ¼ 15),
compared with normoxic (2.85 + 0.40 mmol g21, n ¼ 21)
treated specimens. There was no significant effect of temperature on lactate accumulation in normoxic conditions.
Lactate accumulation was significantly higher at higher
temperatures for hypoxic conditions (ANOVA, f(2,11) ¼
4.92; P , 0.0297, Fig. 4). Lactate accumulation in hypoxia
was an average of 4.51 + 1.23 mmol g21 at 108C, 8.71 +
1.24 mmol g21 at 168 and 17.15 + 4.75 mmol g21 at 208C.
Field study
There was no significant difference in lactate accumulation for specimens collected in the shallow trawl versus
the deep trawl (t-test: t (19) ¼ 21.52; P ¼ 0.1461, Fig. 5,
Table III). CTD data from the day of collection (ETNP
Fig. 3. Routine oxygen consumption rates (MO2) for Phronima
sedentaria, from the Eastern Tropical North Pacific, reported in
micromoles per gram frozen weight per hour on a log scale. MO2 was
significantly related to frozen weight for hypoxic (grey circles) and
normoxic (black squares) treatments. All MO2s were normalized to
208C for comparison and are reported on a log scale. See Table I for
regression equations.
station 2, Fig. 2) were used to determine the oxygen concentrations where specimens were collected. Specimens
from the deep trawl were collected close to or below
P. sedentaria’s critical partial pressure (Pcrit, the oxygen
partial pressure at which an organism’s aerobic metabolic
rate can no longer be maintained, Seibel, 2011) of
28 mM at 108C (Childress, 1975). The shallow trawl
collected specimens at oxygen concentrations above
P. sedentaria’s Pcrit (Table III).
Field caught specimens of P. sedentaria had significantly
higher accumulation of lactate than any of the specimens
used in laboratory experiments (t-test: t (55) ¼ 211.47,
P , 0.001), and a significantly higher lactate accumulation than specimens for normoxia treatment experiments
(t-test: t (40) ¼ 217.30; P , 0.0001, Fig. 5, Table III).
Specimens from the two trawls had a combined average
lactate accumulation of 24.29 + 1.58 mmol g21. The
average for normoxia experiments in the laboratory was
3.60 + 0.67 mmol g21. Specimens from the deep trawl
had an average L-lactate accumulation of 22.56 +
1.38 mmol g21 (n ¼ 10). Shallow trawl specimens had an
average of 26.019 + 1.78 mmol g21 (n ¼ 11).
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M 20.543 and MO2 ¼ 2.4572 M 20.208 for hypoxic and
normoxic treatments, respectively (Fig. 3). The slopes of
hypoxic and normoxic linear regressions were significantly different (ANCOVA : f(2,55) ¼ 34.53; P , 0.0001). For
further comparison, metabolic rates were scaled to a
common weight of 0.15 g using the regression equations
(Table I). Hypoxia had a significant effect on metabolic
rate (t-test: t (56) ¼ 8.25; P , 0.0001; Fig. 3, Supplementary data, Fig. SI).
Normoxic MO2s were normalized to 208C for the
ETNP, Gulf of California and the North Atlantic using
calculated Q10 values when necessary (Table I, Supplementary data, Table SI). Slopes of regression lines for the
three regions are not significantly different (ANCOVA:
f(5,87) ¼ 20.21; P , 0.8103; Table I; Supplementary data,
Fig. S2). There is a significant difference in average MO2
in normoxic conditions between the ETNP, Gulf of California and the North Atlantic (ANCOVA: f(3,89) ¼ 21.88;
P , 0.0001, Table I; Supplementary data, Fig. S2).
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Total metabolism
In P. sedentaria from the ETNP, total metabolism (in ATP
equivalents) was depressed by 78% in the hypoxic experimental conditions, consistent with migration from surface
conditions (normoxia, 208C) to 300 m in the OMZ of
the ETNP (108C, 1% O2). Exposure to OMZ conditions
(108C, 1% O2), compared with normoxic conditions at
the same temperature, caused a 35% reduction in total
metabolism. Surface temperature with OMZ oxygen concentrations (208C, 1% O2) resulted in a 64% reduction in
metabolism compared with normoxic oxygen concentrations at 208C (Fig. 6).
Enzymatic activity
For whole specimens of P. sedentaria, CS activity was
plotted on a log axis to obtain regression equations
(Fig. 7A). The slopes of the linear regressions for each
collection location were significantly different (ANCOVA:
f(5,47) ¼ 14.4, P ¼ ,0.0001) (Fig. 7A). Enzyme activities
were then scaled to a common weight of 0.15 g (using
the regression equations in Fig. 7A, Table IV) to eliminate weight as a factor in the comparison; regressions
could not be compared due to differences in slopes.
There was a significant effect of location on scaled CS
activity, (one-way ANOVA between subjects design,
Fig. 6. Total metabolism of P. sedentaria. Light grey: Adenosine
triphosphate (ATP) produced from anaerobic metabolism, L-lactate mmol
g21. Dark grey: ATP produced from aerobic metabolism, mmol oxygen
g21 h21. At 108C, the combined aerobic and anaerobic ATP production is
reduced by 35% in hypoxic compared with normoxic conditions. At 208C,
total metabolism is reduced by 64% in hypoxic conditions. The migration
from normoxic, 208C conditions to 1% O2, 108C results in a 78%
reduction in total metabolism.
f(2,50) ¼ 30.23; P , 0.0001). Tukey’s honestly significant
difference test showed that specimens from the North
Atlantic had significantly higher CS activity than specimens from the ETNP and California Current (Table IV,
Supplementary data, Fig. S3A; P , 0.05). There were
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Fig. 4. L-Lactate accumulation in whole specimens of Phronima
sedentaria from the Eastern Tropical North Pacific. Light grey: hypoxic,
dark grey: normoxic. Lactate accumulation was significantly higher at
higher temperatures for hypoxic conditions. *, ** and *** indicate that
each temperature is significantly different in hypoxia (P , 0.05). There
was no significant effect of temperature on lactate accumulation in
normoxic conditions. For 108C n ¼ 5 in normoxia and 3 in hypoxia, for
168C n ¼ 10 in normoxia and 9 in hypoxia, for 208C n ¼ 8 in
normoxia and 4 in hypoxia. All values shown are means + SE.
Fig. 5. Lactate accumulation in specimens of Phronima sedentaria,
collected directly from deep (250–300 m) and shallow (25– 50 m)
trawls, compared with experimental organisms subjected to normoxia
at 10 and 208C. There is no significant difference between the deep and
shallow trawls. Oxygen concentration at the depth where specimens
were collected was below P. sedentaria’s critical partial pressure for the
deep trawl. All values are mean + SE. * and ** indicate a significant
difference in lactate accumulation (P , 0.05).
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no significant differences between the ETNP and
California Current.
In ETNP specimens, LDH activities scaled positively
with body mass (Fig. 7B). The slopes of the regressions
were significantly different (one-way ANCOVA: f(5,45) ¼
6.08, P , 0.0002, Fig. 7B). Enzyme activity was then
scaled to a common weight of 0.15 g using the regression
equations from Fig. 7B. There was no significant effect of
location on LDH activity (one-way ANOVA between subjects design: f(2,48) ¼ 2.17; P , 0.1251, Supplementary
data, Fig. S3B).
DISCUSSION
Metabolic rate
The mean MO2 for P. sedentaria, normalized to 208C, is
significantly different between the ETNP, Gulf of
California and North Atlantic (Table I, Supplementary
data, Fig. S2). The average rate for the ETNP is 20%
higher than the Gulf of California. As shown in Tables I
and II, the rates for the ETNP and California Current
are within the range of most literature values. The
sample size for the North Atlantic is small (4 total), a
larger sample size is needed to clarify if rates are higher
in this region. The difference in MO2 between the
ETNP and North Atlantic could be the result of differences in regional productivity at the time of collection.
Extended periods of low food availability can result in
decreased MO2 due to the reduced physiological activity
Table II: Mass-specific rates of oxygen
consumption (mmol O2 g21 h21) from previous
studies for Phronima sedentaria
MO2
(mmol O2 g21 h21) Location
Reference
2.13
2.68
3.65
13.7
Childress, 1975
Ikeda, 2012
Mayzaud, 1973
Bishop and Granger, 2006
California Current
Western Subarctic Pacific
Mediterranean Sea
Central Atlantic
associated with growth, protein synthesis and feeding
(Brockington and Clarke, 2001).
Bishop and Geiger (Bishop and Geiger, 2006) reported
a mean MO2 for P. sedentaria in the Central Atlantic that is
6.4 times higher than other literature values and the rates
from the current study (Table II). Bishop and Geiger’s
rates may be elevated by stress as the specimens were
acclimated to laboratory conditions for 1 h before measurements (inadequate for gut clearance and acclimation
to chambers). Two North Atlantic specimens from the
current study were not used in the analysis because they
were both brooding females and had very high rates,
18.67 and 9.74 mmol g21 h21 at 208C. The higher of
the two was very active in the chamber, and had been
used for photographs prior to incubation, therefore
representing an extremely stressed individual.
Phronima sedentaria’s MO2 (Table I) is close to the relatively low rates of many mesopelagic dwelling organisms
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Fig. 7. (A) Mass-specific activity in units g21 for citrate synthase (CS) and (B) lactate dehydrogenase (LDH) in whole specimens of Phronima sedentaria
shown on a log scale. Regression equations are shown on the graphs. CS is an indicator of aerobic potential and LDH is an indicator on anaerobic
potential. Location has a significant effect on LDH activity, error bars represent standard deviation. The x in both plots represents the mean activity
level from a previous measurement done in the Bahamas, in the Central Atlantic by Bishop and Geiger, 2006, the x-axis error bar represents the
size range for that study, the y-axis error bar represents the range in activity for their study.
L. E. ELDER AND B. A. SEIBEL
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ECOPHYSIOLOGY OF PHRONIMA SEDENTARIA
Table III: Lactate accumulation (mmol g21) and relative hypoxia tolerance in selected crustacean species
Species
Habitat
Callianassa
californiensis
Upogebia
pugettenisis
Phronima sedentaria
Intertidal mud
flat burrows
Intertidal mud
flat burrows
Diel migrator in
OMZ
Euphausia eximia
Diel migrator in
OMZ
Intertidal
Palaemon elegans
Intertidal
Meganyctiphanes
norvegica
Diel migrator in
fjords
Oxygen levels
Hypoxia
tolerant
Reference
21
Lactate
P crit
Anoxia 12 h
0 kPa
11.3 + 0.6 mmol g
Y
Zebe, 1982
Anoxia 12 h
0 kPa
22.1 + 5.6 mmol g21
Y
Zebe, 1982
Deep trawl
Shallow trawl
Hypoxia 108C
Hypoxia 168C
Normoxia
Hypoxia
Normoxia
Hypoxic 108C
1.581 –10.637 mM
48.9 –195.3 mM
0.8 kPa (10 mM)
0.8 kPa
21 kPa
0.8 kPa
21 kPa
0.66 kPa
(8.96 mM)
0 kPa
22.56 + 1.38 mmol g21
26.019 + 1.78 mmol g21
4.51 + 1.24 mmol g21
8.71 + 1.24 mmol g21
2.85 + 0.40 mmol g21
7 mmol g21
2 mmol g21
13.1 + 0.25 mmol g21
Y
This study
Y
Seibel, 2011
N
Taylor and
Spicer, 1987
N
Taylor and
Spicer, 1987
N
Spicer et al.,
1999
21
Anoxia,
immediately
after death
Normoxia
Anoxia,
immediately
after death
Normoxia
Hypoxia
Hypoxia 18 h
21 kPa
0 kPa
3.4 –4.2 mmol g21
9.6 mmol g21
21 kPa
1.8 kPa
6 kPa
Hypoxia 18 h
14.9 kPa
3.4 –4.2 mmol g21
N/A due to 100% mortality
9.91 + 1.68 mmol L21
(haemolymph)
3.01 + 1.05 mmol L21
(haemolymph)
despite its shallow minimum depth of occurrence (MDO;
25 m). Respiratory rates in some mid-water groups decrease with increasing depth of occurrence (Childress,
1975). In mid-water crustaceans from the waters of southern California, excluding P. sedentaria, the range in rate
for epipelagic species (MDO, 0 –100 m) was 3.47 –
21
(Childress, 1975). The
17.32 mmol g21
wet weight (ww) h
range in rate for mesopelagic (MDO 400– 900 m) species
21
in the same study was 0.924 – 2.4 mmol O2 g21
ww h . Low
MO2s in mesopelagic zooplankton are hypothesized to
result from decreasing selection for locomotory capacity
because low light levels limit predator– prey interactions
among visually oriented organisms (Childress, 1995;
Seibel and Drazen, 2007). Phronima sedentaria’s low MO2
may be related to its transparency, as this limits their visibility to predators and prey even in well-lit surface waters.
Cephalopods are highly visual predators that exhibit a
decline in oxygen consumption with increasing
minimum habitat depth similar to crustaceans. However,
squids from the family Cranchiidae have a low MO2
despite occupying shallow water for at least part of their
life history. It has been suggested that transparency
relieves them from selective pressures on locomotion and
metabolism associated with predator– prey interactions
(Seibel and Carlini, 2001). Phronima is highly transparent
(Johnsen, 2001), as is the salp barrel they are housed in.
In fact, hyperiid amphipods are the only group of pelagic
28 mM at
108C
16.7 mmol g
4 –5 kPa
at 88C
arthropods that are truly dominated by transparent
forms (Johnsen, 2001).
L-Lactate
In laboratory experiments, whole specimens of P. sedentaria
exposed to 1% oxygen had a significantly higher accumulation of lactate than specimens exposed to normoxic
conditions (Fig. 4, Table III). Increasing temperature significantly elevated the lactate accumulation in hypoxic
exposed specimens, but did not have a significant effect
on normoxic exposed specimens (Fig. 4). The lactate accumulation in hypoxic conditions is lower than reported
concentrations for other crustaceans considered to be
relatively hypoxia intolerant ((Taylor and Spicer, 1987;
Spicer et al., 1999), Table III). The low levels of lactate
accumulated during hypoxic exposure in P. sedentaria are
possible because total metabolism is depressed, an ability
that many other species apparently lack.
The scope for total lactate production may be correlated with the duration of environmental exposure to
hypoxia or anoxia (Pritchard and Eddy, 1979). The
prawns P. elegans and P. serratus have a low capacity for
lactate accumulation, indicating they cannot survive long
periods of hypoxia. Immediately after death, maximum
lactate concentrations in tissue are 16.7 and 9.6 mmol g21
for P. elegans and P. serratus, respectively ((Taylor and Spicer,
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Palaemon serratus
Conditions
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Total metabolism
When P. sedentaria was exposed to conditions mimicking
their daytime migrations into the OMZ, total metabolism
was depressed by 78% relative to normoxic conditions at
surface temperatures (Fig. 6). Anaerobic metabolism (estimated from lactate accumulation) did increase in hypoxic
conditions, but was not enough to compensate for the decrease in aerobic ATP production during hypoxic exposure.
Hypoxic conditions alone, reduced total metabolism by
35% compared with normoxia at the same temperature.
In pronounced OMZs, where oxygen concentrations
are commonly below 5% of air saturation (1% O2,
15 mM), metabolic depression is anticipated to be a
widespread mechanism allowing energy conservation
during daytime forays into hypoxia (Seibel, 2011). Two
other vertical migrators found in the ETNP exhibit metabolic depression under the same conditions to which
P. sedentaria was subjected (1% O2 at 108C): Humboldt
squid and the euphausiid Euphausia eximia. Humboldt
squid, Dosidicus gigas, reduced total metabolism by 82%
(Rosa and Seibel, 2010). Euphausia eximia exhibits a 45%
reduction in total metabolism (Seibel, 2011). Additional
work in the ETNP has demonstrated metabolic rate
depression in the copepod S. subtenuis (Cass and Daly,
2014); exposed to 3% oxygen at 178C), and three species
of pteropod ((Maas et al., 2012) reduced respiration rate
35– 50% under 1% oxygen at 118C), but the anaerobic
contribution to total metabolism was not measured on
these organisms.
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Metabolic depression below resting metabolic rate
would include reduction of movement, feeding, digestion,
heart rate and ventilation (Storey and Storey, 1990).
Decreased swimming speeds in low oxygen have been
noted for two species of krill (Teal and Carey, 1967;
Klevjer and Kaartvedt, 2011) The diel migrating amphipod Themisto japonica has low locomotory activity during
the day in oxygenated conditions (Hiroki, 1988). One
study demonstrated that P. sedentaria will swim actively
only at low light levels (below 3 cd m22), and suggested
that this is a mechanism to remain at a constant light
level (isolume) and therefore, maintain the desired depth
in the water column (Land, 1992). Thus, it is not possible
to conclude definitively that low oxygen is driving the
reduced locomotion at depth in OMZs. Regardless,
reduced activity in response to low light at depth represents an adaptation that facilitates survival in low oxygen
regions.
Passage of salps through the gut of Phronima at night,
required on average, 4 h 46 min and more than 14 h
during the day (Diebel, 1988). This suggests that P. sedentaria may be able to decrease metabolism by reducing
feeding and digestion rates at depth. In addition, P. sedentaria is able to regulate biochemical pathways to accomplish metabolic rate depression. This is evident because
the current study eliminated feeding and digestion as
factors with a long acclimation period, and movement
was minimized by keeping specimens in darkness.
Therefore, metabolic depression exhibited by hypoxiatreated specimens compared with the control specimens
must have been accomplished by the shutdown of cellular
processes. The arrest of cellular processes as potential
mechanisms for rate reduction has not yet been examined in hyperiid amphipods, but may include reduced
protein synthesis, reduced transcription/translation or
ion transport (reviewed by: Storey and Storey, 2004).
In the OMZ of the California Current, some migrating crustaceans are able to regulate their routine metabolism down to the lowest oxygen level they experience,
and therefore remain aerobic (Antezana, 2002). These
species have very low critical partial pressures (Pcrit), at
which anaerobic metabolic pathways are up-regulated
(Pörtner and Grieshaber, 1993; Seibel, 2011). At oxygen
concentrations below the Pcrit, anaerobic pathways may
be used as a supplement to oxidative phosphorylation for
ATP production. In more pronounced OMZs, such as
the one in the ETNP, it is uncommon for organisms to
remain fully aerobic at depth because the oxygen levels
are below their Pcrit. Seibel (Seibel, 2011) postulated a
hypoxic threshold (0.8 kPa), below which further enhancement of oxygen extraction capacity is constrained.
It is not known if P. sedentaria has adaptations for
enhanced oxygen extraction. Hyperiid amphipods that
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1987), Table III). Anoxia tolerant crustaceans have been
found to have much higher maximum levels of lactate,
up to 60 mmol g21 lactate in burrowing shrimp species
Upogebia pugettenisis ((Zebe, 1982) Table III).
Phronima sedentaria has a relatively high capacity for
lactate accumulation, as shown by the trawl caught specimens (Fig. 5, Table III). There was no significant difference in lactate accumulation for specimens collected in
the shallow versus deep trawl (Fig. 5, Table III). Specimens experienced the stresses of capture in the net
including: crowding, containment, temperature and pressure changes, among others. Experimental organisms
were allowed to recover from capture stress during acclimation, resulting in lower lactate concentrations than
organisms frozen directly from the trawl. This indicates
the importance of laboratory acclimation before conducting physiological experiments. Following exposure below
their Pcrit, the relatively low levels of lactate accumulated
in P. sedentaria from laboratory experiments is consistent
with metabolic depression and minimal requirement for
anaerobic metabolism.
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ECOPHYSIOLOGY OF PHRONIMA SEDENTARIA
Table IV: Citrate synthase and lactate dehydrogenase activity (units g21) and regression equations for
Phronima sedentaria from the three different study locations
Activity (units g21)
Regression equation
Enzyme
Location
N
Size range (g)
Range
Mean
Scaled to 0.15 g
CS
California Current
Eastern Tropical North Pacific
North Atlantic
California Current
Eastern Tropical North Pacific
North Atlantic
21
25
8
20
23
8
0.04 –0.39
0.07 –0.47
0.058 –0.497
0.04 –0.39
0.07 –0.47
0.058 –0.497
0.87–2.94
0.63–1.73
1.02–3.23
5.21–22.3
4.70–38.86
4.79–20.28
1.37 + 0.1
1.11 + 0.07
2.23 + 0.27
9.89 + 1.06
19.002 + 2.09
9.96 + 1.73
1.25 + 0.07
1.05 + 0.06
2.133 + 0.16
11.08 + 1.67
13.87 + 1.47
9.89 + 1.67
LDH
Enzymatic activity
The metabolic enzyme CS is an indicator of aerobic potential and LDH is an indicator of anaerobic glycolytic potential. Both of these enzymes have been previously measured
in Phronima specimens from Exumas Sound, Bahamas
(Bishop and Geiger, 2006) where there is no OMZ. The
average CS activity of P. sedentaria from the Bahamas was
3.00 + 1.90 units g21 (mean size 0.25 g, range 0.04–
0.45 g). The CS activity of P. sedentaria in the Bahamas is
higher than the mean activity for all three locations used in
this study (Table IV, Fig. 7B), but is within the range of
values reported here. This difference may be an artefact of
the size distribution of the specimens used by Bishop and
Geiger, for which we have only the range. The size ranges
for their study and ours overlap but if the distribution is
skewed toward large or small specimens, the mean enzymatic activity will be similarly skewed.
Specimens from the North Atlantic had a significantly
higher CS activity than the other two locations (Table IV,
Fig. 7A). Nutritional status contributes to differences in
metabolic enzyme activities in copepods, with activity decreasing in unfed specimens (Clarke and Walsh, 1993).
Similarly, CS activity in the hepatopancreas of two deep
sea crabs was significantly lower after 1 month of food
deprivation, although activity in muscle tissue was not
affected (Company et al., 2008). CS activity in the North
Atlantic was 0.68 units g21 higher than that measured in
the ETNP and California Current. The higher aerobic
capacity is consistent with the higher average metabolic
rate in the North Atlantic than the other locations
(Supplementary data, Fig. S2). The higher CS activity in
the North Atlantic specimens, and the Bishop and
Geiger study could be due to differences in food availability in the regions when the studies were conducted.
Gonzalez and Quiñones (Gonzalez and Quiñones,
2002) hypothesized that LDH activity would be elevated
in organisms adapted to low oxygen environments.
Evidence in the literature for increased LDH activity in
organisms, particularly crustaceans, adapted to hypoxia
is mixed. Epipelagic copepods have a lower LDH activity,
and are therefore less reliant on glycolytic energy sources
than mesopelagic and bathypelagic copepods. Meso- and
bathypelagic copepods may use glycolysis as an energy
source for burst swimming in low oxygen (Thuesen et al.,
1998). Thuesen et al. hypothesize that survival in low
oxygen is influenced by buffering ability and substrate
stores and that LDH is primarily for burst swimming
(Thuesen et al., 1998).
High LDH activities in some medusae were hypothesized to help sustain swimming during vertical migration,
and also promote hypoxia tolerance when migrating
through OMZs (Thuesen et al., 2005). In the Humboldt
current system off South America, where there is a permanent subsurface OMZ, the euphausiid, Euphasia mucronata, has a LDH activity two orders of magnitude higher
than the copepod, Calanus chilensis (Gonzalez and
Quiñones, 2002). Calanus chilensis is a non-migrator that
remains in oxygenated waters and is much smaller in
maximum body size than the vertically migrating
E. mucronata. Given that C. chilensis and E. mucronata are
not only different taxa, but also ecologically distinct, this
comparison does little to answer the question at hand. To
test the hypothesis of elevated LDH activity relating to
survival in hypoxia, the same, or closely related species,
should be compared from regions with and without
OMZs. This type of comparison would avoid confusion
from variation in ecology and life history.
The LDH activity of P. sedentaria from the Bahamas
measured at 208C was 3.00 + 2.00 units g21 (mean size
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have been examined do not have oxygen binding pigments to enhance oxygen extraction from the water
(Spicer and Morritt, 1995). The reported mean Pcrit for
P. sedentaria in the California Current is 2.11 kPa (28 mM
at 108C (Childress, 1975)). For this study, individuals
from the ETNP were able to survive 6 h at 0.8 kPa at
108C (13.4 mM) but accumulated 4.51 + 1.23 mmol g21
lactate. Assuming the Pcrit is the same for the ETNP as
the California Current, P. sedentaria is adapted to survive
below its Pcrit by depressing total metabolism and increasing anaerobic metabolism.
CS ¼ 0.811x 20.214
CS ¼ 1.3609x 0.157
CS ¼ 1.1204x 20.328
LDH ¼ 24.63x 0.443
LDH ¼ 49.073x 0.727
LDH ¼ 7.341x 20.108
JOURNAL OF PLANKTON RESEARCH
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Body size in relation to oxygen availability
Chapelle and Peck (Chapelle and Peck, 2004) proposed
that the concentration of oxygen in the water limits the
maximum potential size in aquatic amphipods. Spicer
and Gaston (Spicer and Gaston, 1999) argued that
oxygen partial pressure, not its concentration, would determine the restrictions on size (see Supplementary
Material for further discussion). The current study did
not set out to address the ongoing debate of how oxygen
concentration, partial pressure or a combination of the
two, drives patterns in body size of aquatic ectotherms.
However, if oxygen concentration is the limiting factor in
maximum body size, then this trend would also be seen
across gradients of the water column such as in OMZs.
We collected a single species of amphipod from four different locations, each with varying oxygen concentrations; from a severe OMZ in the ETNP to no OMZ in
the North Atlantic (Fig. 2). Due to this broad coverage,
we felt it relevant to address the ongoing debate by examining the size range of specimens from our collection.
There was no significant difference in body size
between the four study sites (one-way ANOVA f(3,5) ¼
4.31; P , 0.0748 comparing the 95% largest specimens,
see Supplementary Material for more details). The lack
of a significant difference in maximum size between locations indicates environmental oxygen concentration does
not limit maximum size in this amphipod.
Significance
Climate change is causing an increase in surface water
temperature and decrease in oxygen concentrations
(Keeling et al., 2010), which will have important impacts
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on zooplankton ecology, vertical distribution and physiology, as well as carbon cycling in the region (Vinogradov
and Voronina, 1962; Seibel, 2011). Ecological implications include: altered species composition of an area,
changes in prey availability, prey size or predation risk
and/or changes in trophic dynamics due to shifts in
predator– prey interactions (Taylor and Rand, 2003;
Kodama et al., 2006; Ikeda, 2012; Wishner et al., 2013).
Anaerobic metabolism and metabolic depression are
not sustainable for long periods of time due to substrate
limitation and end-product accumulation. Organisms
must return to oxygenated surface waters for part of the
night to burn off accumulated end products. As surface
waters warm, they will become stressful for some organisms, restricting how shallow zooplankton migrate (Seibel,
2011; Elder and Seibel, 2015). Phronima sedentaria occasionally experiences its upper critical temperature of 298C
during the summer in the ETNP (Fig. 2A), and demonstrates a thermal stress response when exposed to this temperature (Elder and Seibel, 2015). This stress response
includes oxygen limitation in normoxic waters due to a
mismatch between oxygen supply and demand at higher
temperatures. Other diel migrators are likely to live close
to their thermal limits. Increasing temperature and decreasing oxygen supply will vertically compress the habitable night-time depth range of diel migrating species, both
from above and below (see Fig. 7 in (Seibel, 2011).
In the southern California Current region, a .60%
decline in some mesopelagic fishes is likely due to the
decline of mid-water oxygen levels. The aggregation of
mesoplagic micronekton in the hypoxic boundary layer
of the OMZ in the California Current, suggests that they
descend as deeply as possible to evade visual predators
while avoiding the effects of hypoxia. The shoaling of the
OMZ may increase the vulnerability of these diel migrators by forcing them into better-lit waters during the day,
enhancing the chance of predation from visually oriented
predators (Koslow et al., 2011). Expanding OMZs would
similarly affect zooplankton diel migrators that track
oxygen levels (Wishner et al., 2013).
Diel migrators that are not able to alter daytime
depths will be exposed to lower oxygen for a greater time
and distance. In the ETNP, the daytime biomass peak at
200 – 300 m, associated with diel vertical migration and
located at the upper oxycline or OMZ core, was present
at the same depth at two locations, despite different
oxygen concentrations between the locations (Wishner
et al., 2013). Nordic krill, Meganyctiphanes norvegica, is an
example of a crustacean that is not specifically adapted to
maintain oxygen uptake or capacity for anaerobic metabolism, but still vertically migrates into hypoxia (oxygen
concentrations equivalent to their Pcrit of 4 – 6 kPa). Their
migration rhythm must be strong, and not overridden by
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0.25 g, range 0.04 – 0.45 g, (Bishop and Geiger,
2006), which is lower than activities for all locations in
this study (Fig. 7B). Similar to the difference in CS activity between the present study and Bishop and Geiger, the
lower LDH value may be an artefact of the size distribution of the specimens, or variation in nutritional status.
Phronima sedentaria is expected to use anaerobic glycolysis
for burst swimming as well as metabolic demand while
migrating into regions of low oxygen. Anaerobic glycolysis may be an important strategy for burst swimming
when manoeuvring the salp barrel they live in (Bishop
and Geiger, 2006). In the current study, P. sedentaria mean
scaled LDH activity for a 0.15-g organism measured at
208C was not significantly different between specimens
collected from regions with OMZs versus the oxygenated
Atlantic Ocean (Supplementary data, Fig. S3B). This
study adds to the growing support that LDH activity is
not related to survival in low oxygen environments.
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L. E. ELDER AND B. A. SEIBEL
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ECOPHYSIOLOGY OF PHRONIMA SEDENTARIA
low oxygen stress, since these krill enter potentially lethal
conditions presumably to avoid visual predators (Spicer
et al., 1999). As OMZs expand, some species of zooplankton may not be able to modify this migration rhythm.
The distance to travel and duration in low oxygen could
be beyond their physiological abilities, which could
compromise their long-term existence in regions with
expanding OMZs (Wishner et al., 2013).
Diel migrating zooplankton play a significant role in
the biological carbon pump (Ducklow et al., 2001).
Zooplankton consume phytoplankton near the ocean
surface at night and migrate down during the day where
they metabolize ingested food, release carbon in the
forms of dissolved organic carbon, sinking faecal material
and CO2, therefore transporting carbon to depth
(Longhurst et al., 1990; Ducklow et al., 2001). Respiration
and metabolic activity are among the most important
components of carbon flux (Burd et al., 2010). To depress
metabolism, P. sedentaria will decrease feeding, digestion
and respiration. This depression will result in a reduction
of faecal pellet production and CO2 excretion at depth,
leading to an overall decrease in the species’ contribution
to carbon flux. If metabolic depression is common to
vertically migrating zooplankton, the decreased carbon
input at depth would reduce the efficiency of the biological carbon pump in regions with pronounced OMZs
(Seibel, 2011).
S U P P L E M E N TA RY DATA
CONCLUSIONS
FUNDING
In the ETNP, the amphipod P. sedentaria is adapted for diel
exposure to critical oxygen partial pressures by depressing
metabolism while migrating into the OMZ. LDH activity
of P. sedentaria did not increase with decreasing environmental oxygen concentrations. This indicates that the
enzyme LDH is not used to increase anaerobic potential
for P. sedentaria to survive migration into hypoxic conditions. As global warming continues, OMZs are predicted
to expand and P. sedentaria may change its vertical depth
range to avoid hypoxic waters and thermal stress at the
surface. This would have significant impacts on predator–
prey interactions in the region as well as carbon cycling
(Seibel, 2011). Metabolic depression may be a common
adaptation in OMZ dwelling zooplankton (Seibel, 2011);
therefore, OMZ expansion will have a similar effect on
night-time habitat ranges of other diel migrators.
Anaerobic metabolism and metabolic depression are not
sustainable for long periods of time due to substrate limitations and end-product accumulation. Therefore, the longterm existence of some species of zooplankton may be
compromised in OMZs if the distance to travel and duration in low oxygen are beyond physiological abilities.
This work was supported by the following National
Science Foundation grants: In the ETNP OCE-0526502
to K.W. and B.S. In the North Atlantic OCE-0852160
and in the Gulf of California OCE-0526493, both to
B.S. Support was awarded to L.E. for work in the
California Current as a participant in the 2012
University-National Oceanographic Laboratory System
(UNOLS) chief scientist training cruise, which was
funded by National Science Foundation grant
OCE-1041068.
Supplementary data can be found online at http://plankt.
oxfordjournals.org.
AC K N OW L E D G E M E N T S
REFERENCES
Antezana, T. (2002) Adaptive behaviour of Euphausia mucronata in relation to the oxygen minimum layer of the Humboldt Current.
Oceanogr. East. Pac., 2, 29– 40.
Beecham, R. V., Small, B. C. and Minchew, C. D. (2006) Using portable
lactate and glucose meters for catfish research: acceptable alternatives
to established laboratory methods? North Am. J. Aquacult., 68,
291 –295.
Bergmeyer, H. U., Bergmeyer, J. and Grabl, M. (1985) Methods of
Enzymatic Analysis. Verlag Chemie, Weinheim, Germany, VIII.
909
Downloaded from http://plankt.oxfordjournals.org/ at Yale University on September 22, 2015
Thanks to Kendra Daly for organizing the ETNP
cruises, which provided ship time for L. E. (National
Science Foundation grant OCE-0526545). Thanks to
Karen Wishner for critical review on earlier drafts of this
paper, as well as insightful discussions. Useful comments
by two anonymous reviewers also improved the quality of
this manuscript. This research would not have been possible without the Captains and crews of the R/V Knorr,
R/V New Horizon, R/V Endeavor and R/V Steward
Johnson. Thanks also to R. Rosa, T. Towanda,
J. Schneider, C. Cass, L. Trueblood, S. Bush, B. Phillips,
A. Maas and A. Nyack for assistance in net deployment
for specimen collection. The Bongo net used for specimen collection during the California Current Cruise was
loaned to L.E. from the Pelagic Invertebrates Collection
of Scripps Institute of Oceanography. Thanks to Mark
Ohman and Shonna Dovel for assistance with bongo net
loan and deployment logistics. Thanks to Clare Reimers
the principal investigator for the chief scientist training
cruise for the opportunely.
JOURNAL OF PLANKTON RESEARCH
j
VOLUME 37
Bishop, R. E. and Geiger, S. P. (2006) Phronima energetics: is there a
bonus to the barrel? Crustaceana, 79, 1059–1070.
Brockington, S. and Clarke, A. (2001) The relative influence of temperature and food on the metabolism of a marine invertebrate. J. Exp.
Mar. Biol. Ecol., 258, 87– 99.
Brooks, G. (2002) Lactate shuttles in nature. Biochem. Soc. Trans., 30,
258–264.
Burd, A. B., Hansell, D. A., Steinberg, D. K., Anderson, T. R.,
Arı́stegui, J., Baltar, F., Beaupre, S. R., Buesseler, K. O. et al. (2010)
Assessing the apparent imbalance between geochemical and biochemical indicators of meso-and bathypelagic biological activity:
what the@ $]! is wrong with present calculations of carbon budgets?
Deep Sea Res. Part II, 57, 1557– 1571.
j
NUMBER 5
j
PAGES 897 – 911
j
2015
Elder, L. E. and Seibel, B. A. (2015) The thermal stress response to diel
vertical migration in the hyperiid amphipod Phronima sedentaria. Comp.
Biochem. Physiol., 187, 20– 26.
Engel, P. C. and Jones, J. B. (1978) Causes and elimination of erratic blanks
in enzymatic metabolite assays involving the use of NADþ in alkaline
hydrazine buffers: improved conditions for the assay of L-glutamate,
L-lactate, and other metabolites. Anal. Biochem., 88, 475–484.
Fernández-Álamo, M. A. and Färber-Lorda, J. (2006) Zooplankton and
the oceanography of the eastern tropical Pacific: a review. Prog.
Oceanogr., 69, 318–359.
Fiedler, P. C. and Talley, L. D. (2006) Hydrography of the eastern tropical Pacific: a review. Prog. Oceanogr., 69, 143–180.
Foucreau, N., Cottin, D., Piscart, C. and Hervant, F. (2013)
Physiological and metabolic responses to rising temperature in
Gammarus pulex (Crustacea) populations living under continental or
Mediterranean climates. Comp. Biochem. Physiol., 168, 69–75.
Chapelle, G. and Peck, L. S. (2004) Amphipod crustacean size spectra:
new insights in the relationship between size and oxygen. Oikos, 106,
167–175.
Gonzalez, R. R. and Quiñones, R. A. (2002) LDH activity in Euphausia
mucronata and Calanus chilensis: implications for vertical migration behaviour. J. Plankton Res., 24, 1349– 1356.
Childress, J. J. (1975) The respiratory rates of midwater crustaceans
as a function of depth of occurrence and relation to the oxygen
minimum layer off southern California. Comp. Biochem. Physiol., 50,
787–799.
Guppy, M. and Withers, P. (1999) Metabolic depression in animals:
physiological perspectives and biochemical generalizations. Biol. Rev.
Camb. Philos. Soc., 74, 1– 40.
Childress, J. J. (1977) Effects of pressure, temperature and oxygen on the
oxygen consumption rate of the midwater copepod Gaussia princeps.
Mar. Biol., 39, 19–24.
Gutmann, I. and Wahlefeld, A. W. (1974) L-(þ)-Lactate determination
with lactate dehydrogenase and NAD. In Bergmeyer, H. U. (ed.),
Methods of Enzymatic Analysis, 3rd edn. Academic Press, New York.
Childress, J. J. (1995) Are there physiological and biochemical adaptations
of metabolism in deep-sea animals? Trends Ecol. Evol., 10, 30–36.
Hiroki, M. (1988) Relation between diel vertical migration and locomotor activity of a marine hyperiidean amphipod, Themisto japonica
(Bovallius). J. Crustacean Biol., 8, 48–52.
Childress, J. J., Barnes, A. T., Quetin, L. B. and Robison, B. (1978)
Thermally protecting cod ends for the recovery of living deep-sea
animals. Deep Sea Res., 25, 419–422.
Hirose, E., Aoki, M. N. and Nishikawa, J. (2005) Still alive? Fine
structure of the barrels made by Phronima (Crustacea: Amphipoda).
J. Mar. Biol. Assoc. UK, 85, 1435– 1439.
Childress, J. J. and Mickel, T. J. (1980) A motion compensated shipboard precision balance system. Deep Sea Res., 27, 965–970.
Hochachka, P. W. and Somero, G. N. (2002) Biochemical Adaptation:
Mechanism and Process in Physiological Evolution. Oxford University Press,
USA.
Childress, J. J. and Seibel, B. A. (1998) Life at stable low oxygen levels:
adaptations of animals to oceanic oxygen minimum layers. J. Exp.
Biol., 201, 1223–1232.
Clark, L. C. (1956) Monitor and control of blood and tissue oxygen tensions. Trans. Am. Soc. Artif. Intern. Organs, 2, 41–48.
Clarke, M. E. and Walsh, P. J. (1993) Effect of nutritional status on
citrate synthase activity in Acartia tonsa and Temora longicornis. Limnol.
Oceanogr., 38, 414– 418.
Company, J. B., Thuesen, E. V., Childress, J. J., Rotllant, G. and Zal, F.
(2008) Effects of food deprivation on enzymatic activities of the
Mediterranean deep-sea crab, Geryon longipes A. Milne-Edwards, 1882
and the Pacific hydrothermal vent crab, Bythograea thermydron Williams,
1980 (Decapoda, Brachyura). Crustaceana, 81, 67–85.
Deutsch, C., Brix, H., Ito, T., Frenzel, H. and Thompson, L. A. (2011)
Climate-forced variability of ocean hypoxia. Science, 333, 336– 339.
Diebel, C. E. (1988) Observations on the anatomy and behavior of
Phronima sedentaria (forskal) (amphipod: hyperiidea). J. Crust. Biol., 8,
79– 90.
Doney, S. C., Ruckelshaus, M., Duffy, J. E., Barry, J. P., Chan, F.,
English, C. A., Galindo, H. M., Grebmeier, J. M. et al. (2012)
Climate change impacts on marine ecosystems. Ann. Rev. Mar. Sci., 4,
11– 37.
Ducklow, H. W., Steinberg, D. K. and Buesseler, K. O. (2001) Upper
ocean carbon export and the biological pump. Oceanography, 14,
50– 58.
Ikeda, T. (2012) Metabolism and chemical composition of zooplankton
from 500 to 5,000 m depth of the western subarctic Pacific Ocean.
J. Oceanogr., 68, 641– 649.
Johnsen, S. (2001) Hidden in plain sight: the ecology and physiology of
organismal transparency. Biol. Bull., 201, 301– 318.
Kamykowski, D. and Zentara, S.-J. (1990) Hypoxia in the world ocean
as recorded in the historical data set. Deep Sea Res. Part A, 37,
1861–1874.
Keeling, R. F. and Garcia, H. E. (2002) The change in oceanic O2 inventory associated with recent global warming. Proc. Natl. Acad. Sci.
USA, 99, 7848– 7853.
Keeling, R. F., Körtzinger, A. and Gruber, N. (2010) Ocean deoxygenation in a warming world. Ann. Rev. Mar. Sci., 2, 199–229.
Klevjer, T. A. and Kaartvedt, S. (2011) Krill (Meganyctiphanes norvegica)
swim faster at night. Limnol. Oceanogr., 56, 765–774.
Kodama, K., Horiguchi, T., Kume, G., Nagayama, S., Shimizu, T.,
Shiraishi, H., Morita, M. and Shimizu, M. (2006) Effects of hypoxia
on early life history of the stomatopod Oratosquilla oratoria in a
coastal sea. Mar. Ecol. Prog. Ser., 324, 197 –206.
Koslow, J., Goericke, R., Lara-Lopez, A. and Watson, W. (2011) Impact
of declining intermediate-water oxygen on deepwater fishes in the
California Current. Mar. Ecol. Prog. Ser., 436, 207–218.
Land, M. F. (1992) Locomotion and visual behaviour of mid-water crustaceans. J. Mar. Biol. Assoc. UK, 72, 41–60.
910
Downloaded from http://plankt.oxfordjournals.org/ at Yale University on September 22, 2015
Cass, C. J. and Daly, K. L. (2014) Eucalanoid copepod metabolic rates
in the oxygen minimum zone of the eastern tropical north Pacific:
effects of oxygen and temperature. Deep Sea Res. Part I, 94, 137– 149.
L. E. ELDER AND B. A. SEIBEL
j
ECOPHYSIOLOGY OF PHRONIMA SEDENTARIA
Laval, P. (1978) The barrel of the pelagic amphipod Phronima sedentaria
(Forsk.) (crustaces: Hyperiidea). J. Exp. Mar. Biol. Ecol., 33, 187–211.
Shih, C.-T. (1969) The systematics and biology of the family
Phronimidae (Crustacea: Amphipoda). Dana Reports no. 74, 1 –100.
Longhurst, A. R., Bedo, A. W., Harrison, W. G., Head, E. J. H. and
Sameoto, D. D. (1990) Vertical flux of respiratory carbon by oceanic
diel migrant biota. Deep Sea Res. Part A, 37, 685–694.
Shulenberger, E. (1977) Hyperiid amphipods from the zooplankton
community of the North Pacific central gyre. Mar. Biol., 42,
375 – 385.
Maas, A. E., Wishner, K. F. and Seibel, B. A. (2012) Metabolic suppression
in thecosomatous pteropods as an effect of low temperature and hypoxia
in the eastern tropical North Pacific. Mar. Biol., 159, 1955–1967.
Spicer, J. I. and Gaston, K. J. (1999) Amphipod gigantism dictated by
oxygen availability? Ecol. Lett., 2, 397 –403.
Madin, L. P. and Harbison, G. R. (1977) The associations of Amphipoda
Hyperiidea with gelatinous zooplankton—I. associations with Salpidae.
Deep Sea Res., 24, 449 –463.
Marsh, A. G. and Manahan, D. (1999) A method for accurate measurements of the respiration rates of marine invertebrate embryos and
larvae. Mar. Ecol. Prog. Ser., 184, 1 –10.
Mcdonald, D. G., Mcfarlane, W. J. and Milligan, C. L. (1998)
Anaerobic capacity and swim performance of juvenile salmonids.
Can. J. Fish. Aquat. Sci., 55, 1198–1207.
Packard, T. T., Minas, H. J., Coste, B., Martinez, R., Bonin, M. C.,
Gostan, J., Garfield, P., Christensen, J. et al. (1988) Formation of the
Alboran oxygen minimum zone. Deep Sea Res. Part A, 35, 1111– 1118.
Pérez, E. H., Dawood, H., Chetty, U., Esterhuizen, T. M. and Bizaare,
M. (2008) Validation of the Accutrendw lactate meter for hyperlactatemia screening during antiretroviral therapy in a resource-poor
setting. Int. J. Infect. Dis., 12, 553–556.
Pörtner, H. O. and Grieshaber, M. K. (1993) Critical Po2(s) in oxyconforming and oxyregulation animals: gas exchange, metabolic rate
and the mode of energy production. In Bicudo, J. E. P. W. (ed.), The
Vertebrate gas Transport Cascade: Adaptations to Environment and Mode of Life,
CRC Press, Boco Raton FL, pp. 330–357.
Pritchard, A. and Eddy, S. (1979) Lactate formation in Callianassa californiensis and Upogebia pugettensis (Crustacea: Thalassinidea). Mar. Biol.,
50, 249–253.
Robinson, C., Steinberg, D. K., Anderson, T. R., Arı́stegui, J., Carlson,
C. A., Frost, J. R., Ghiglione, J.-F., Hernández-León, S. et al. (2010)
Mesopelagic zone ecology and biogeochemistry –a synthesis. Deep Sea
Res. Part II Top. Stud. Oceanogr., 57, 1504–1518.
Rosa, R. and Seibel, B. A. (2010) Metabolic physiology of the
Humboldt squid, Dosidicus gigas: implications for vertical migration in
a pronounced oxygen minimum zone. Prog. Oceanogr., 86, 72–80.
Seibel, B. A. (2011) Critical oxygen levels and metabolic suppression in
oceanic oxygen minimum zones. J. Exp. Biol., 214, 326–336.
Spicer, J. I., Thomasson, M. A. and Strömberg, J. O. (1999) Possessing a
poor anaerobic capacity does not prevent the diet vertical migration
of Nordic krill Meganyctiphanes norvegica into hypoxic waters. Mar. Ecol.
Prog. Ser., 185, 181– 187.
Storey, K. B. and Storey, J. M. (1990) Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation.
Q. Rev. Biol., 65, 145– 174.
Stramma, L., Johnson, G. C., Sprintall, J. and Mohrholz, V. (2008)
Expanding oxygen-minimum zones in the tropical oceans. Science,
320, 655.
Taylor, A. C. and Spicer, J. I. (1987) Metabolic responses of the prawns
Palaemon elegans and P. serratus (Crustacea: Decapoda) to acute hypoxia
and anoxia. Mar. Biol., 95, 521– 530.
Taylor, J. C. and Rand, P. S. (2003) Spatial overlap and distribution of
anchovies (Anchoa spp.) and copepods in a shallow stratified estuary.
Aquat. Living Resour., 16, 191–196.
Teal, J. M. and Carey, F. G. (1967) Respiration of a euphausiid from the
oxygen minimum layer. Limnol. Oceanogr., 12, 548– 550.
Thuesen, E. V., Mccullough, K. D. and Childress, J. J. (2005) Metabolic
enzyme activities in swimming muscle of medusae: is the scaling of
glycolytic activity related to oxygen availability? J. Mar. Biol. Assoc.
UK, 85, 603–611.
Thuesen, E. V., Miller, C. B. and Childress, J. J. (1998) Ecophysiological
interpretation of oxygen consumption rates and enzymatic activities
of deep-sea copepods. Mar. Ecol. Prog. Ser., 168, 95–107.
Vinogradov, M. and Voronina, N. (1962) Influence of the oxygen deficit
on the distribution of plankton in the Arabian Sea. Deep Sea Res.
Oceanogr. Abstr. Elsevier, 9, 523–530.
Vinogradov, M. E., Volkov, A. F., Semenova, T. N. and Siegel-Causey,
D. (1996) Hyperiid Amphipods (Amphipoda, Hyperiidea) of the World Oceans.
Science Publications Incorporated, Lebanon, USA.
Wishner, K., Levin, L., Gowing, M. and Mullineaux, L. (1990)
Involvement of the oxygen minimum in benthic zonation on a deep
seamount. Nature, 346, 57– 59.
Seibel, B. A. and Carlini, D. B. (2001) Metabolism of pelagic cephalopods
as a function of habitat depth: a reanalysis using phylogenetically independent contrasts. Biol. Bull. Mar. Biol. Lab. Woods Hole, 201, 1–5.
Wishner, K. F., Outram, D. M., Seibel, B. A., Daly, K. L. and Williams,
R. L. (2013) Zooplankton in the Eastern Tropical North Pacific:
boundary effects of oxygen minimum zone expansion. Deep Sea Res.
Part I, 79, 122–140.
Seibel, B. A. and Drazen, J. C. (2007) The rate of metabolism in marine
animals: environmental constraints, ecological demands and energetic opportunities. Philos. Trans. R. Soc. B Biol. Sci., 362, 2061.
Zebe, E. (1982) Anaerobic metabolism in Upogebia pugettensis and
Callianassa californiensis(crustacea, thalassinidea). Comp. Biochem. Physiol.,
72, 613 –617.
911
Downloaded from http://plankt.oxfordjournals.org/ at Yale University on September 22, 2015
Mayzaud, P. (1973) Respiration and nitrogen excretion of zooplankton.
II. Studies of the metabolic characteristics of starved animals. Mar.
Biol., 21, 19– 28.
Spicer, J. I. and Morritt, D. (1995) Oxygen carriage by the haemolymph
of hyperiid amphipods. J. Mar. Biol. Assoc. UK, 75, 997– 998.